Method for determining hematocrit corrected analyte concentrations

ABSTRACT

Description is provided herein for an embodiment of a method determining a hematocrit-corrected glucose concentration. The exemplary method includes providing a test strip having a reference electrode and a working electrode, wherein the working electrode includes a plurality of microelectrodes and is coated with at least an enzyme and a mediator. The method can be achieved by: providing a test strip comprising a reference electrode and a working electrode formed with a plurality of microelectrodes and coated with a reagent layer; applying a fluid sample to the test strip for a reaction period; applying a test voltage between the reference electrode and the working electrode; measuring a test current as a function of time; measuring a steady state current value when the test current has reached an equilibrium; calculating a ratio of the test current to the steady state current value; plotting the ratio of the test current to the steady state current value as a function of the inverse square root of time; calculating an effective diffusion coefficient from the slope of the linearly regressed plot of the ratio of the test current to the steady state current value as a function of the inverse square root of time; and calculating a hematocrit-corrected concentration of analyte.

1. PRIORITY

This application claims the benefits of priority under 35 U.S.C. §119from provisional application Ser. No. 60/850,173 filed on Oct. 5, 2006,entitled: “Method for Determining Hematocrit Corrected AnalyteConcentrations,” which application is incorporated by reference in itsentirety herein.

2. BACKGROUND OF THE INVENTION

Electrochemical glucose test strips, such as those used in the OneTouch®Ultra® whole blood testing kit, which is available from LifeScan, Inc.,are designed to measure the concentration of glucose in a blood samplefrom patients with diabetes. The measurement of glucose is based uponthe specific oxidation of glucose by the flavo-enzyme glucose oxidase.The reactions which may occur in a glucose test strip are summarizedbelow in Equations 1 and 2.

D-Glucose+GO_((ox)→Gluconic Acid+GO) _((red))  (1)

GO_((red))+2Fe(CN)₆ ³⁻→GO_((ox)+)2Fe(CN)₆ ⁴⁻  (2)

As shown in Equation 1, glucose is oxidized to gluconic acid by theoxidized form of glucose oxidase (GO_((ox)). It should be noted thatGO_((ox)) may also be referred to as an “oxidized enzyme”. During thereaction in Equation 1, the oxidized enzyme GO_((ox)) is converted toits reduced state which is denoted as GO_((red)) (i.e., “reducedenzyme”). Next, the reduced enzyme GO_((red)) is re-oxidized back toGO_((ox)) by reaction with Fe(CN)₆ ³⁻ (referred to as either theoxidized mediator or ferricyanide) as shown in Equation 2. During there-generation of GO_((ox)) back to its oxidized state GO(CN)₆ ³⁻ isreduced to Fe(CN)₆ ⁴⁻ (referred to as either reduced mediator orferrocyanide).

When the reactions set forth above are conducted with a test voltageapplied between two electrodes, a test current may be created by theelectrochemical re-oxidation of the reduced mediator at the electrodesurface. Thus, since, in an ideal environment, the amount offerrocyanide created during the chemical reaction described above isdirectly proportional to the amount of glucose in the sample positionedbetween the electrodes, the test current generated would be proportionalto the glucose content of the sample. A mediator, such as ferricyanide,is a compound that accepts electrons from an enzyme such as glucoseoxidase and then donates the electrons to an electrode. As theconcentration of glucose in the sample increases, the amount of reducedmediator formed also increases, hence, there is a direct relationshipbetween the test current resulting from the re-oxidation of reducedmediator and glucose concentration. In particular, the transfer ofelectrons across the electrical interface results in a flow of testcurrent (2 moles of electrons for every mole of glucose that isoxidized). The test current resulting from the introduction of glucosemay, therefore, be referred to as a glucose current.

Because it can be very important to know the concentration of glucose inblood, particularly in people with diabetes, test meters have beendeveloped using the principals set forth above to enable the averageperson to sample and test their blood to determine the glucoseconcentration at any given time. The glucose current generated ismonitored by the test meter and converted into a reading of glucoseconcentration using an algorithm that relates the test current to aglucose concentration via a simple mathematical formula. In general, thetest meters work in conjunction with a disposable test strip thatincludes a sample receiving chamber and at least two electrodes disposedwithin the sample receiving chamber in addition to the enzyme (e.g.glucose oxidase) and the mediator (e.g. ferricyanide). In use, the userpricks their finger or other convenient site to induce bleeding andintroduces a blood sample to the sample receiving chamber, thus startingthe chemical reaction set forth above.

In electrochemical terms, the function of the meter is two fold.Firstly, it provides a polarizing voltage (approximately 0.4 V in thecase of OneTouch® Ultra®) that polarizes the electrical interface andallows current flow at the carbon working electrode surface. Secondly,it measures the current that flows in the external circuit between theanode (working electrode) and the cathode (reference electrode). Thetest meter may, therefore be considered to be a simple electrochemicalsystem that operates in a two-electrode mode although, in practice,third and, even fourth electrodes may be used to facilitate themeasurement of glucose and/or perform other functions in the meter.

In most situations, the equation set forth above is considered to be asufficient approximation of the chemical reaction taking place on thetest strip and the test meter outputting a sufficiently accuraterepresentation of the glucose content of the blood sample. However,under certain circumstances and for certain purposes, it may beadvantageous to improve the accuracy of the measurement. For example,blood samples having a high hematocrit level or low hematocrit level maycause a glucose measurement to be inaccurate.

A hematocrit level represents a percentage of the volume of a wholeblood sample occupied by red blood cells. The hematocrit level may alsobe represented as a fraction of red blood cells present in a whole bloodsample. In general, a high hematocrit blood sample is more viscous (upto about 10 centipoise at 70% hematocrit) than a low hematocrit bloodsample (about 3 centipoise at 20% hematocrit). In addition, a highhematocrit blood sample has a higher oxygen content than low hematocritblood because of the concomitant increase in hemoglobin, which is acarrier for oxygen. Thus, the hematocrit level can influence theviscosity and oxygen content of blood. As will be later described, bothviscosity and oxygen content may change the magnitude of the glucosecurrent and in turn cause the glucose concentration to be inaccurate.

A high viscosity sample (i.e., high hematocrit blood sample) can causethe test current to decrease for a variety of factors such as a decreasein 1) the dissolution rate of enzyme and/or mediator, 2) the enzymereaction rate, and 3) the diffusion of a reduced mediator towards theworking electrode. A decrease in current that is not based on a decreasein glucose concentration can potentially cause an inaccurate glucoseconcentration to be measured.

A slower dissolution rate of the reagent layer can slow down theenzymatic reaction as shown in Equations 1 and 2 because the oxidizedenzyme GO_((ox)) must dissolve first before it can react with glucose.Similarly, ferricyanide (Fe(CN)₆ ³⁻) must dissolve first before it canreact with reduced enzyme GO_((red)). If the undissolved oxidized enzymeGO_((ox)) cannot oxidize glucose, then the reduced enzyme GO_((red))cannot produce the reduced mediator Fe(CN)₆ ⁴⁻ needed to generate thetest current. Further, oxidized enzyme GO_((ox)) will react with glucoseand oxidized mediator Fe(CN)₆ ³⁻ more slowly if it is in a highviscosity sample as opposed to a low viscosity sample. The slowerreaction rate with high viscosity samples is ascribed to an overalldecrease in mass diffusion. Both oxidized enzyme GO_((ox)) and glucosemust collide and interact together for the reaction to occur as shown inEquation 1. The ability of oxidized enzyme GO_((ox)) glucose to collideand interact together is slowed down when they are in a viscous sample.Yet further, reduced mediator Fe(CN)₆ ⁴⁻ will diffuse to the workingelectrode slower when dissolved in a high viscosity sample. Because thetest current is typically limited by the diffusion of reduced mediatorFe(CN)₆ ⁴⁻ to the working electrode, a high viscosity sample will alsoattenuate the test current. In summary, there are several factors thatcause the test current to decrease when the sample has an increasedviscosity.

A high oxygen content may also cause a decrease in the test current. Thereduced enzyme (GO_((red))) can reduce oxygen (O₂) to hydrogen peroxideas shown be Equation 3.

GO_((red))+O₂→GO_((ox))+H₂O₂  (3)

As noted earlier, the reduced enzyme GO_((red)) can also reduceferricyanide (Fe(CN)₆ ³⁻) to ferrocyanide (Fe(CN)₆ ⁴⁻) as shown inEquation 2. Thus, oxygen can compete with ferricyanide for reacting withthe reduced enzyme (GO_((red))). In other words, the occurrence of thereaction in Equation 3 will likely cause a decrease in the rate of thereaction in Equation 2. Because of such a competition betweenferricyanide and oxygen, a higher oxygen content will cause lessferrocyanide to be produced. In turn, a decrease in ferrocyanide wouldcause a decrease in the magnitude of the test current. Therefore, a highoxygen content blood sample can potentially decrease the test currentand affect the accuracy of the glucose measurement.

As such, applicants have great interest in the development of methodsreducing the effects of hematocrit on a glucose measurement. In certainprotocols, a pre-cast blood filtering membrane that is separate from thereagent layer has been employed to remove red blood cells and therebyreduce the hematocrit effect. The pre-cast blood filtering membranewhich is separated from the reagent layer can be disposed on the workingelectrode. The use of a discrete pre-cast blood filtering membrane isunsatisfactory in that it requires a more complex test strip, increasedsample volume, and increased testing time. The blood filtering membraneretains a certain amount of blood that does not contact the workingelectrodes causing a need for a larger blood sample. In addition, afinite amount of time is needed for the blood to be filtered by themembrane causing an increase in the overall test times. Thus, applicantsrecognize that it would be advantageous to reduce the effects ofhematocrit without using a pre-cast blood filtering membrane that isseparate from the reagent layer.

In the prior art, the hematocrit effect may be reduced by applyingmultiple test voltages such as, for example, a sinusoidal test voltage.However, applying a sinusoidal test voltage results in a more complexand expensive test meter. Further, the test meter needs to measure thetest currents accurately and precisely at pre-determined time intervals.The electronic components can be expensive and complicated for a testmeter to accurately and precisely apply multiple test voltages.

Applicants realize that it would be advantageous to implement a systemhaving a test meter that applies only one test voltage and a test stripthat does not use a pre-cast membrane to reduce the effects ofhematocrit. The system instead uses a test strip having a workingelectrode with a plurality of microelectrodes formed thereon. Moreparticularly, applicants recognizes that it would be advantageous todevelop an algorithm that mathematically processes the collected testcurrent using one test voltage such that an accurate glucoseconcentration can be determined that reduces the effects of hematocrit.

Furthermore, applicants have determined that it would be beneficial toprovide a mechanism whereby the test meter can differentiate between abodily fluid, for example whole blood, and a control solution.Similarly, it would be beneficial to provide a method whereby a testmeter can determine if a test strip includes a plurality ofmicroelectrodes formed on a working electrode.

SUMMARY OF THE INVENTION

In one aspect, a method is provided for determining ahematocrit-corrected glucose concentration. The exemplary methodincludes providing a test strip having a reference electrode and aworking electrode, wherein the working electrode includes a plurality ofmicroelectrodes and is coated with at least an enzyme and a mediator.The method can be achieved by: providing a test strip comprising areference electrode and a working electrode formed with a plurality ofmicroelectrodes and coated with a reagent layer; applying a fluid sampleto the test strip for a reaction period; applying a test voltage betweenthe reference electrode and the working electrode; measuring a testcurrent as a function of time; measuring a steady state current valuewhen the test current has reached an equilibrium; calculating a ratio ofthe test current to the steady state current value; plotting the ratioof the test current to the steady state current value as a function ofthe inverse square root of time; calculating an effective diffusioncoefficient from the slope of the linearly regressed plot of the ratioof the test current to the steady state current value as a function ofthe inverse square root of time; and calculating a hematocrit-correctedconcentration of analyte.

In another aspect of the present invention, the exemplary method furtherincludes steps for distinguishing between a bodily fluid and a controlsolution. The method includes comparing the calculated value of theeffective diffusion coefficient to an acceptance range for either abodily fluid or a control solution, depending on the sample applied tothe test strip. If the calculated value is not within the acceptancerange for bodily fluid or control solution, the test meter will notallow the user to proceed with testing and will display an appropriateerror message.

In another aspect, a method of determining a type of fluid sampleapplied to the test strip is provided. The method can be achieved by:providing a test strip having a reference electrode and a workingelectrode, wherein the working electrode is formed with a plurality ofmicroelectrodes and is coated with a reagent layer; applying a fluidsample to the test strip for a reaction period; applying a test voltagebetween the reference electrode and the working electrode; measuring atest current as a function of time; measuring a steady state currentvalue when the test current has reached an equilibrium; calculating aratio of the test current to the steady state current value; plottingthe ratio of the test current to the steady state current value as afunction of the inverse square root of time; calculating an effectivediffusion coefficient from the slope of the linearly regressed plot ofthe ratio of the test current to the steady state current value as afunction of the inverse square root of time; determining a type of afluid sample applied to the test strip by comparing a measured value forthe effective diffusion coefficient against an acceptance range for abodily fluid and a control solution; and displaying an appropriate errormessage if the effective diffusion coefficient does not pass theacceptance range for the type of fluid sample applied to the test strip.

In yet another aspect of the present invention, the exemplary methodfurther includes steps for determining if a test strip includes amicroelectrode array. The method includes using the effective diffusioncoefficient to calculate a temperature-corrected effective diffusioncoefficient. The calculated value for the temperature-correctedeffective diffusion coefficient is then compared to an acceptance rangefor a test strip that includes a plurality of microelectrodes. If thecalculated value is within the acceptance range for test strips having aplurality of microelectrodes, the user may proceed with testing.However, if the calculated value is not within the acceptance range fortest strips having a plurality of microelectrodes, an appropriate errormessage is displayed on the test meter and the test meter will not allowthe user to proceed with testing.

BRIEF DESCRIPTION OF DRAWINGS

A better understanding of the features and advantages of the presentinvention will be obtained by reference to the following detaileddescription that sets forth illustrative embodiments, in which theprinciples of the invention are utilized, and the accompanying drawingsof which:

FIG. 1 is a top exploded perspective view of an unassembled test stripaccording to an exemplary embodiment of the present invention;

FIG. 2 is a top view of the test strip as shown in FIG. 1 after it hasbeen assembled;

FIGS. 3-7 are top views of a distal portion of a partially assembledtest strip according to exemplary embodiments of the present invention;

FIG. 8 is a cross sectional view of the test strip shown in FIG. 3through a microelectrode array on a first working electrode according toan exemplary embodiment;

FIG. 9 is a cross sectional view through a microelectrode array on afirst working electrode of FIG. 3 with additional layers coated on aninsulation portion including a reagent layer, adhesive pads, and ahydrophilic portion. The reagent layer is disposed on the distal side ofthe hydrophilic portion according to an exemplary embodiment;

FIG. 10 is a cross sectional view through a microelectrode array on afirst working electrode of FIG. 3 with additional layers coated on aninsulation portion including a reagent layer, adhesive pads, and ahydrophilic portion. The reagent layer is disposed over the insulationportion according to an exemplary embodiment;

FIG. 11 is a top, close up view of the plurality of microelectrodes onthe first working electrode of the test strip shown in FIG. 3 accordingto an exemplary embodiment;

FIG. 12 is a top view of a test meter connected to the test strip ofFIGS. 1 and 2;

FIG. 13 is a simplified schematic view of the test meter of FIG. 12forming an electrical connection with the test strip of FIGS. 1 and 2;

FIGS. 14 and 15 are graphical representations of a test voltage appliedto a working electrode of a test strip according to methods of thepresent invention;

FIG. 16 is a plot of current as a function of time (i.e., a current-timetransient) for a fluid sample which is generated by a method;

FIG. 17 is a plot of

$\frac{I(t)}{I_{\;}}$

as a function of

$\frac{1}{\sqrt{t}}$

which is generated by a method;

FIG. 18 is a flowchart illustrating a sequence of steps in a method todetermine a hematocrit-corrected analyte concentration reported by atest meter according to an exemplary embodiment;

FIG. 19 is a flowchart illustrating a sequence of steps in a method todetermine whether a bodily fluid or a control solution has been added toa test strip according to an exemplary embodiment;

FIG. 20 is a flowchart illustrating a sequence of steps in a method todetermine if a test strip includes a microelectrode array according toan exemplary embodiment;

FIG. 21 is a plot illustrating simulated data used to differentiatebetween a test strip that includes a microelectrode array and a teststrip that does not include a microelectrode array, wherein the plot isgenerated by a method; and

FIGS. 22 and 23 are perspective and side views, respectively, of amedical device that is suitable for use in the present invention.

DETAILED DESCRIPTION OF THE EXEMPLARY EMBODIMENTS

The disclosure below describes the measurement of a glucoseconcentration in a whole blood sample; however, the person of ordinaryskill will recognize that the description is readily adapted to measurethe properties of other analytes, such as cholesterol, ketone bodies oralcohol, and to other fluids such as saliva, urine, interstitial fluid,or test strip control solutions.

It will be further understood that this invention is not limited to onlycorrecting for hematocrit and can also be applicable to correcting forthe effect of variable viscosity or oxygen content in fluid samples. Forexample, whole blood samples can have a high viscosity for a variety ofother reasons in addition to high hematocrit including low temperature(e.g., about 10° C.), high lipid concentration, and/or high proteinconcentration.

It will yet further be understood that the invention would also beapplicable for reducing the effects caused by oxygen and/or viscosity ofphysiological fluids other than blood. For example, physiological fluidsmay also include plasma, serum, interstitial fluid, and a combinationthereof. It should be noted that it is not uncommon for extractedinterstitial fluid samples to be partially mixed with blood.

The following sections will describe a test strip embodiment that may beused with an algorithm according to one embodiment for calculating ahematocrit-corrected glucose concentration. FIG. 1 is an explodedperspective view of a test strip 100, which includes multiple layersdisposed upon a substrate 5. These layers may include a conductive layer50, an insulation layer 16, a reagent layer 22, an adhesive layer 60, ahydrophilic layer 70, and a top layer 80. Test strip 100 may bemanufactured in a series of steps wherein conductive layer 50,insulation layer 16, reagent layer 22 and adhesive layer 60 aresequentially deposited on substrate 5 using, for example, a screenprinting process as described in U.S. Pre-Grant Publication No.US20050096409A1 and published International Application No.'sWO2004040948A1, WO2004040290A1, WO2004040287A1, WO2004040285A2,WO2004040005A1, WO2004039897A2, and WO2004039600A2. In an alternativeembodiment, an ink jetting process may be used to deposit reagent layer22 on substrate 5. An example ink jetting process is described in U.S.Pat. No. 6,179,979. Hydrophilic layer 70 and top layer 80 may bedisposed from a roll stock and laminated onto substrate 5. Test strip100 also includes a distal portion 3 and a proximal portion 4 as shownin FIGS. 1 and 2.

The fully assembled test strip 100, as shown in FIG. 2, includes aninlet 82 through which a blood sample may be drawn into a samplereceiving chamber 84. Inlet 82 may be formed by cutting through a distalportion 3 of test strip 100. A blood sample 94 can be applied to inlet82 to fill a sample receiving chamber 84 so that glucose can bemeasured, as shown in FIG. 12. The side edges of a first adhesive pad 24and a second adhesive pad 26 located adjacent to reagent layer 22 eachdefine a wall of sample receiving chamber 84. A bottom portion or“floor” of sample receiving chamber 84 includes a portion of substrate5, conductive layer 50, and insulation layer 16. A top portion or “roof”of sample receiving chamber 84 includes distal hydrophilic portion 32.

For test strip 100, as shown in FIG. 1, conductive layer 50 includes areference electrode 10, a first working electrode 12, a second workingelectrode 14, a first contact 13, a second contact 15, a referencecontact 11, and a strip detection bar 17. Suitable materials which maybe used for the conductive layer are Au, Pd, Ir, Pt, Rh, stainlesssteel, doped tin oxide, carbon, and the like. In one embodiment, thematerial for the conductive layer may be a carbon ink such as thosedescribed in U.S. Pat. No. 5,653,918. In another embodiment, thematerial for the conductive layer may be a sputtered metal such as goldor palladium. A laser ablated pattern may be formed into the sputteredmetal layer to form a plurality of electrodes.

For test strip 100, insulation layer 16 includes first aperture 18 whichexposes a portion of reference electrode 10, openings 20 which expose aportion of first working electrode 12, and second aperture 21 whichexposes a portion of second working electrode 14. The portions ofreference electrode 10, first working electrode 12 and second workingelectrode 14 exposed by first aperture 18, openings 20 and secondaperture 21, respectively, can be wetted by a liquid sample as shown inFIG. 1. Openings 20 in insulation layer expose a plurality ofmicroelectrodes 120 as will be described with reference to FIGS. 5-9. Inone exemplary embodiment, insulation layer 16 is Ercon E6110-116 JetBlack Insulayer™ ink that may be purchased from Ercon, Inc (Waltham,Mass.).

Reagent layer 22 may be disposed on a portion of conductive layer 50,substrate 5, and insulation layer 16 as shown in FIG. 1. In anembodiment, reagent layer 22 may include an enzyme, a mediator thatselectivity reacts with glucose and a buffer for maintaining a desiredpH. Examples of enzymes suitable for use in this invention may includeeither glucose oxidase or glucose dehydrogenase. More specifically, theglucose dehydrogenase may have a pyrroloquinoline quinone co-factor(abbreviated as PQQ or may be referred to its common name which ismethoxatin). Examples of mediator suitable for use in this invention mayinclude either ferricyanide or ruthenium hexamine (Ru^(III)(NH₃)₆).During the reactions as shown in Equations 1 and 2, a proportionalamount of reduced mediator can be generated that is electrochemicallymeasured for calculating a glucose concentration. Examples of bufferssuitable for use in the present invention may include phosphate, citrateor citraconate. Examples of reagent formulations or inks suitable foruse in the present invention can be found in U.S. Pat. Nos. 5,708,247and 6,046,051 and published international applications WO01/67099 andWO01/73124.

In an embodiment, the formulation may include a 200 millimolar phosphatebuffer having a pH of about 7 and a ruthenium hexamine mediator. The pHof around 7 was chosen because glucose oxidase has a sufficiently highactivity at this pH when using ruthenium hexamine as a mediator. In anembodiment, the formulation may have an enzyme activity ranging fromabout 1500 units/mL to about 8000 units/mL. The enzyme activity rangemay be selected so that the glucose current does not depend on the levelof enzyme activity in the formulation so long as the enzyme activitylevel is within the above stated range. The enzyme activity should besufficiently large to ensure that the resulting glucose current will notbe dependent on small variations in the enzyme activity. For instance,the glucose current will depend on the amount of enzyme activity in theformulation if the enzyme activity is less than 1500 units/mL. On theother hand, for enzyme activity levels greater than 8000 units/mL,solubility issues may arise where the glucose oxidase cannot besufficiently dissolved in the formulation. Glucose oxidase may becommercially available from Biozyme Laboratories International Limited(San Diego, Calif., U.S.A.). The glucose oxidase may have an enzymeactivity of about 250 units/mg where the enzyme activity units are basedon an o-dianisidine assay at pH 7 and 25° C.

Optionally, reagent layer 22 includes a matrix material that aides inretaining the reagent layer 22 on the surface of conductive layer 50 inthe presence of fluid sample. Useful matrix materials include silicassuch as Cab-o-Sil® TS630 or Cab-o-Sil® 530 (Cabot Corporation, Boston,USA). While not wishing to be bound by any particular theory, it isbelieved that silica forms a gel network in the presence of the samplethat effectively maintains the coating on the surface of the electrode.Other useful matrix materials include polymeric materials such aspolyethersulfones, acrylic and methacrylic acid polymers; polymersderived from starch, cellulose and other natural polysaccharides;polyamides and collagen. An example of a useful coating composition isdisclosed in Example 1 of U.S. Pat. No. 5,708,247. Reagent layer 22 mayalso optionally include at least one stabilizing agent such as albumin,sucrose, trehalose, mannitol or lactose, an agent such ashydroxyethylcellulose to adjust the viscosity, an antifoam agent such asDC1500, and at least one wetting agent such as polyvinylpyrrilidone orpolyvinyl acetate.

For test strip 100, adhesive layer 60 includes first adhesive pad 24,second adhesive pad 26, and third adhesive pad 28 as shown in FIG. 1. Inan embodiment, adhesive layer 60 may comprise a water based acryliccopolymer pressure sensitive adhesive, which is commercially availablefrom Tape Specialties LTD (Tring, Herts, United Kingdom; part#A6435).Adhesive layer 60 is disposed on a portion of insulation layer 16,conductive layer 50, and substrate 5. Adhesive layer 60 bindshydrophilic layer 70 to test strip 100.

Hydrophilic layer 70 includes a distal hydrophilic portion 32 andproximal hydrophilic portion 34. As a non-limiting example, hydrophiliclayer 70 is a polyester having one hydrophilic surface such as ananti-fog coating which is commercially available from 3M.

For test strip 100, top layer 80 includes a clear portion 36 and opaqueportion 38 as shown in FIG. 1. Top layer 80 is disposed on and adheredto hydrophilic layer 70. In one embodiment, top layer 80 is a polyester.It should be noted that the clear portion 36 substantially overlapsdistal hydrophilic portion 32 that allows a user to visually confirmthat the sample receiving chamber 84 is sufficiently filled. Opaqueportion 38 helps the user observe a high degree of contrast between acolored fluid such as, for example, blood within the sample receivingchamber 84 and the opaque portion 38 of top layer 80.

FIG. 3 shows a simplified top view of a partial assembly of a test strip100 that includes a first working electrode 12 in the form of amicroelectrode array 110 according to an exemplary embodiment. Ingeneral, microelectrode array 110 will enhance the effects of radialdiffusion causing an increase in the measured current density (currentper unit area of the working electrode). Radial diffusion refers to theflux of reduced mediator that diffuses to first working electrode 106 ina non-perpendicular manner with respect to a plane of first workingelectrode 106. In contrast, planar diffusion refers to the flux ofreduced mediator that diffuses to first working electrode 106 in anapproximately perpendicular manner with respect to a plane of firstworking electrode 106. As a result of the enhanced radial diffusion, theapplication of a limiting test voltage to microelectrode array 110 cancause a test current to achieve a non-zero steady-state value that isindependent of time. In contrast, the application of a limiting testvoltage to a non-microelectrode will result in a test current thatapproaches zero as time progresses. Because the steady-state value isindependent of time for a microelectrode array 110, an effectivediffusion coefficient D of the mediator in the blood sample may becalculated. In turn, effective diffusion coefficient D can be used as aninput into an algorithm for reducing the effects of hematocrit.

Referring again to FIG. 3, a distal portion 102 of test strip 100includes a reference electrode 10, a first working electrode 12 and asecond working electrode 14. First working electrode 10 is in the formof a microelectrode array 110 that includes a plurality ofmicroelectrodes 120.

In another embodiment shown in FIG. 4, a distal portion 202 of a teststrip 200 includes a reference electrode 204, a first working electrode206 and a second working electrode 208. Test strip 200 differs from teststrip 100 in that a second working electrode 208 includes amicroelectrode array 210 with a plurality of microelectrodes 220. In yetanother embodiment shown in FIG. 5, both a first working electrode 306and a second working electrode 308 include a microelectrode array 310with a plurality of microelectrodes 320.

Another embodiment of a test strip 400 having a microelectrode array 410is shown in FIG. 6. A distal portion 402 of test strip 400 includes areference electrode 404, a working electrode 406 and a fill detectelectrode 412. Working electrode 406 is in the form of a microelectrodearray 410 that includes a plurality of microelectrodes 420. Test strip400 differs from test strip 100 in that working electrode 406 is locatedupstream of reference electrode 404 and does not include a secondworking electrode. As shown in FIG. 6, working electrode 406 mayoptionally be at least twice the surface area of reference electrode404.

FIG. 7 shows yet another embodiment of a test strip 500 in which teststrip 500 includes a reference electrode 504 and a working electrode506. In this embodiment, reference electrode 504 and working electrode506 are of approximately equal surface area. Working electrode 506includes a microelectrode array 510 and is downstream from referenceelectrode 504 with respect to fluid flow in test strip 500. FIG. 7 alsoshows an optional fill detect electrode 512.

Many of the layers of test strip 100, as shown in FIG. 1, may be usedfor test strips 100, 200, 300, 400 and 500, such as insulation layer 16,reagent layer 22, adhesive layer 60, hydrophilic layer 70, and top layer80.

FIG. 8 is a cross-sectional view through microelectrode array 110 onfirst working electrode 106 of FIG. 3 showing that an insulation portion130 is disposed on first working electrode 106. In one exemplaryembodiment, insulation portion 130 is contiguous with insulation layer16 of FIG. 1. Thus, in this embodiment, insulation portion 130 isprinted in the same step as the printing of insulation layer 16. Laserablating insulation portion 130 to form openings 20 which expose aplurality of microelectrodes 120 may then form microelectrode array 110.

In another embodiment, insulation portion 130 is a separate element frominsulation layer 16 of FIG. 1. In this embodiment, insulation portion130 is disposed on first working electrode 106 in a step separate fromthe printing of insulation layer 16. Insulation portion 130 may bedisposed over and bound to first working electrode 106 by processes suchas ultrasonic welding, screen-printing, or through the use of anadhesive. In this embodiment, openings 20 in insulation portion 130 maybe formed before or after adhering insulation portion 130 to firstworking electrode 106.

FIGS. 9 and 10 are cross-sectional views through microelectrode array110 on first working electrode 106 of FIG. 3 with additional layersincluding reagent layer 22, adhesive pads 24 and 26, and hydrophilicportion 32. Reagent layer 22 may be disposed on distal hydrophilicportion 32 as shown in FIG. 9. In this embodiment, reagent layer 22 ishighly soluble, ensuring that when a fluid sample is applied to teststrip 100, reagent layer 22 readily dissolves and the mediator quicklydiffuses to microelectrodes 120. Alternatively, reagent layer 22 may bedisposed over an insulation portion 130 as shown in FIG. 10. In thisembodiment, reagent layer 22 may be insoluble because of the closeproximity of reagent layer 22 to microelectrodes 120. First and secondadhesive pads 24 and 26 are applied in such a manner as to define a gapheight L between insulator portion 130 and reagent layer 22 (see FIG. 9)or between reagent layer 22 and distal hydrophilic portion (see FIG.10). First and second adhesive pads 24 and 26 also define a width W ofreagent layer 22.

For microelectrode array 110 to have an enhanced effect due to radialdiffusion, insulation portion 130 should have the appropriatedimensions. In one aspect, insulation portion 130 may include a height Hthat is between about 1 microns and about 6 microns. It is necessarythat insulation portion 130 be sufficiently thin so as to allow radialdiffusion. If insulation portion 130 is much greater than 6 microns,then insulation portion 130 interferes with radial diffusion and wouldactually promote planar diffusion.

In another aspect shown in FIG. 11, each microelectrode 120 should bespaced sufficiently far from each other so as to prevent a firstmicroelectrode from competing with an adjacent second microelectrode foroxidizing mediator. Each microelectrode 120 may be spaced apart with adistance B ranging from about 5 times to about 10 times the diameter ofmicroelectrode 120. In one embodiment as shown in FIG. 10, eachmicroelectrode 120 may be evenly spaced throughout insulation portion130, where a microelectrode may have six neighboring microelectrodeswhich form a hexagonal shape.

In yet another aspect, each microelectrode 120 should be sufficientlysmall such that the proportion of the test current ascribed to radialdiffusion is greater than the proportion of the test current ascribed toplanar diffusion. Microelectrode 120 may have a disk shape with adiameter A ranging from about 3 microns to about 20 microns. Inalternative embodiments, microelectrode 120 may be square, rectangular,elliptical or oval in shape.

In another aspect, microelectrode array 110 may be any geometric shapeincluding, but not limited to, a circle, an oval, a square or arectangle. If rectangular in shape, the surface area is generallybetween about 0.3 and about 3 square microns.

FIG. 12 illustrates a test meter 600 suitable for connecting to teststrip 100. Test meter 600 includes a display 602, a housing 604, aplurality of user interface buttons 606, and a strip port connector 608.Test meter 600 further includes electronic circuitry within housing 604such as a memory 620, a microprocessor 622, electronic components forapplying a test voltage, and also for measuring a plurality of testcurrent values. Proximal portion 4 of test strip 100 may be insertedinto strip port connector 608. Display 102 may output a glucoseconcentration and be used to show a user interface for prompting a useron how to perform a test. The plurality of user interface buttons 606allow a user to operate test meter 600 by navigating through the userinterface software.

FIG. 13 shows a simplified schematic of a test meter 600 interfacingwith test strip 100. Test meter 600 includes a first connector 612,second connector 614, and a reference connector 610 which respectivelyform an electrical connection to first contact 13, second contact 15,and reference contact 11. The three aforementioned connectors are partof strip port connector 608. When performing a test, a first testvoltage source 616 may apply a first test voltage V₁ between firstworking electrode 12 and reference electrode 10. As a result of firsttest voltage V₁, test meter 600 may then measure a first test currentI₁. In a similar manner, second test voltage source 618 applies a secondtest voltage V₂ between second working electrode 14 and referenceelectrode 10. As a result of second test voltage V₂, test meter 600 maythen measure a second test current I₂. In an embodiment, first testvoltage V₁ and second test voltage V₂ may be about equal allowing aglucose measurement to be performed twice where a first measurement isperformed with first working electrode 12 and a second measurement isperformed with second working electrode 14. The use of two glucosemeasurements can increase accuracy by averaging the two resultstogether. For simplifying the description of the following sections, thealgorithms for determining a hematocrit corrected glucose concentrationwill be described for only one working electrode and referenceelectrode. It should be apparent to one skilled in the art that theinvention should not be limited to one working electrode and referenceelectrode, but that multiple working electrodes may also be applied tothe present invention.

The following methods will describe algorithms that may be applied tomicroelectrodes and, more particularly, to microelectrode arrays, wherethe test current achieves a steady-state value because of a higherproportion of radial diffusion.

FIG. 18 is a flowchart illustrating a sequence of steps in a method 700used by test meter 600 to apply a hematocrit correction to a glucoseconcentration according to an exemplary embodiment.

Method 700 includes providing a test strip 100 with a referenceelectrode 10, a first working electrode 12, an optional second electrode14 and a test meter 600, as set forth by step 710. First workingelectrode 12 includes a plurality of microelectrodes 120 (i.e.,microelectrode array 110) with each disk shaped microelectrode having adiameter of about 3 microns to about 50 microns and separated by about 5to about 10 times the diameter thereof. Reference electrode 10 includesa surface area that is at least equal to the surface area ofmicroelectrode array 110.

FIG. 14 is a graphical representation of a test voltage applied to teststrip 100 according to the method. Before a fluid sample is applied totest strip 100, test meter 600 is in a fluid detection mode in which atest voltage (not shown) of about 100 millivolts to about 600millivolts, typically 400 millivolts is applied between first workingelectrode 12 and reference electrode 10. As set forth in step 720, thefluid sample is applied to test strip 100 at t₀ and is allowed to reactwith reagent layer 22 for a reaction period t_(R). The presence ofsample in the reaction zone of test strip 100 is determined by measuringthe current flowing through first working electrode 12. The beginning ofreaction period t_(R) is determined to begin when the current flowingthrough first working electrode 12 reaches a desired value, typicallyabout 0.150 nanoamperes (not shown), at which point a test voltage ofzero millivolts is applied between first working electrode 12 andreference electrode 10. Reaction period t_(R) is typically between about2 and about 3 seconds and is more typically about 2.5 seconds. Afterreaction period t_(R), the test voltage in the exemplary method isapplied to test strip 100 at t₁ for a total test time t_(T). In analternative method shown in FIG. 15, the reaction period t_(R) isomitted such that the start of the test commences as soon as sufficientcurrent is flowing through first working electrode 12.

As set forth in step 730, a limiting test voltage of about 100millivolts to about 600 millivolts, typically 400 millivolts, is appliedbetween reference electrode 10 and first working electrode 12 and a testcurrent is measured as a function of time, as illustrated in FIG. 16.Note that the test current approaches a steady-state current value astime progresses. A steady state current value I_(SS) is measured when anequilibrium current value is attained, as set forth by step 740. Steadystate current value I_(SS) generally is reached between about 0.5 andabout 2 seconds after the test voltage is applied to test strip 100.

As set forth in step 750, the ratio of the test current to a steadystate current value

$\frac{I(t)}{I_{ss}}$

is then calculated for each time point at which test current ismeasured. For microelectrode array 110 having a plurality of disk-shapedmicroelectrodes 120 where a limiting test voltage is applied, thefollowing equation estimates a ratio of the test current to thesteady-state current value:

$\begin{matrix}{\frac{I(t)}{I_{ss}} = {1 + \left( \frac{2r_{d}}{\pi \sqrt{\pi \; {Dt}}} \right)}} & (4)\end{matrix}$

Where:

I(t) is the test current in microamperes measured at time t;

I_(SS) is the steady-state current value in microamperes;

r_(d) is the radius of microelectode 120 in centimeters;

t is time in seconds; and

D is the effective diffusion coefficient in units of centimeter²/second.

The effective diffusion coefficient D takes into account the diffusionof the mediator in a blood sample having a dissolved reagent layer. Ingeneral, the effective diffusion coefficient D should decrease withincreasing hematocrit levels. Thus, the effective diffusion coefficientD is dependent on the hematocrit level and can be used in an algorithmfor decreasing the effects of hematocrit. The following will describehow to calculate the effective diffusion coefficient D and then applythe effective diffusion coefficient D for calculating a glucoseconcentration.

Using Equation 4, effective diffusion coefficient D may be calculated byplotting the values

$\frac{I(t)}{I_{ss}}$

on the y-axis and

$\frac{1}{\sqrt{t}}$

on the x-axis as illustrated in FIG. 17 and set forth in step 760. Theresulting slope from the linear portion of the line may then becalculated and converted into effective diffusion coefficient D, as setforth in step 770. In practice, all the calculations required todetermine effective diffusion coefficient D would be pre-programmed intothe test meter as an algorithm. An advantage of this approach ofdetermining effective diffusion coefficient D is that effectivediffusion coefficient D is independent of glucose concentration.

As set forth in step 780, effective diffusion coefficient D may be usedwith Equation 5 below to estimate the reduced mediator concentrationC_(red) (e.g., concentration of Fe(CN)₆ ⁴⁻).

$\begin{matrix}{C_{red} = \frac{I_{ss}}{4{nFDr}_{d}}} & (5)\end{matrix}$

Where:

n is the number of electrons exchanged per reduced mediator molecule;

F is Faraday's constant.

C_(red) can then be used to estimate the hematocrit-corrected glucoseconcentration. For example, a calibration curve may be generated inwhich the y-axis is C_(red) where C_(red) is calculated for whole bloodsamples with a range of glucose and hematocrit concentrations. Thex-axis is the reference glucose concentration G_(ref) of the same wholeblood samples as measured on a reference glucose analyzer. Thecalibration intercept may be subtracted from C_(red) followed by adivision using a calibration slope to yield glucose concentrationG_(ref). In summary, Equations 1 and 2 allow for glucose concentrationsto be calculated with a reduced effect from hematocrit when usingmicroelectrode arrays as illustrated in FIGS. 3 to 7, thus resulting ina more accurate glucose concentration.

Lastly, the hematocrit-corrected analyte concentration is displayed ontest meter 600, as set forth in step 790.

FIG. 19 is a flowchart illustrating a sequence of steps in a method 800used by test meter 600 to establish whether a fluid sample is a bodilyfluid (e.g., whole blood) or a control solution according to anexemplary embodiment. A control solution is used to ensure that the testmeter and test strip are functioning properly. In one embodiment,analyte concentrations for a bodily fluid are averaged over a period oftime to assess the patient's health. If a control solution value isstored in the test meter as an analyte concentration, the averageanalyte concentration for bodily fluid will be incorrect. Thus, having amethod to distinguish between a bodily fluid and a control solution isadvantageous.

Method 800 includes providing a test strip 100 with a referenceelectrode 10, a first working electrode 12, an optional second electrode14 and a test meter 600, as set forth by step 810. First workingelectrode 12 includes a plurality of microelectrodes 120 (i.e.,microelectrode array 110) with each disk shaped microelectrode having adiameter of about 3 to about 50 microns and separated by about 5 toabout 10 times the diameter thereof. Reference electrode 10 includes asurface area that is at least equal to the surface area ofmicroelectrode array 110.

As set forth in step 820, the fluid sample is applied to test strip 100at t_(o) and is allowed to react with reagent layer 22 for a reactionperiod t_(R) (see FIG. 14). The presence of sample in the reaction zoneof test strip 100 is determined by measuring the current flowing throughfirst working electrode 12. The beginning of reaction period t_(R) isdetermined to begin when the current flowing through first workingelectrode 12 reaches a desired value, typically about 0.150 nanoamperes(not shown), at which point a test voltage of between about −50millivolts and about +50 millivolts, typically about zero millivolts, isapplied between first working electrode 12 and reference electrode 10.Reaction period t_(R) is typically between about 2 and about 3 secondsand is more typically about 2.5 seconds. After reaction period t_(R),the test voltage in the exemplary method is applied to test strip 100 att_(i) for a total test time t_(T). In an alternative method shown inFIG. 15, the reaction period t_(R) is omitted such that the start of thetest commences as soon as sufficient current is flowing through firstworking electrode 12.

As set forth in step 830, a limiting test voltage of about 100millivolts to about 600 millivolts, typically 400 millivolts, is appliedbetween reference electrode 10 and first working electrode 12 and testcurrent is measured as a function of time, as illustrated in FIG. 16. Asteady state current value I_(SS) is measured when an equilibriumcurrent value is attained, as set forth by step 840. Steady statecurrent value I_(SS) generally is reached between about 0.5 and about 2seconds after the test voltage is applied to test strip 100.

As set forth in step 850, the ratio of the test current to a steadystate current value

$\frac{I(t)}{I_{ss}}$

is then calculated for each time point at which test current ismeasured. For microelectrode array 110 having a plurality of disk-shapedmicroelectrodes 120 where a limiting test voltage is applied, Equation 4above is used to estimate a ratio of the test current to thesteady-state current value.

Using Equation 4, effective diffusion coefficient D may also becalculated by plotting the values

$\frac{I(t)}{I_{SS}}$

on the y-axis and

$\frac{1}{\sqrt{t}}$

on the x-axis as illustrated in FIG. 17 and set forth in step 860. Theresulting slope from the linear portion of the line may then becalculated and converted into effective diffusion coefficient D, as setforth in step 870.

As set forth in step 880, effective diffusion coefficient D may be usedwith Equation 5 above to estimate the reduced mediator concentrationC_(red) (e.g., concentration of Fe(CN)₆ ⁴⁻).

To determine the type of fluid sample (e.g., bodily fluid or controlsolution) applied to test strip 100, test meter 600 compares a measuredvalue for effective diffusion coefficient D to an acceptance range forbodily fluid and an acceptance range for control solution, as set forthby step 880. For whole blood having a hematocrit level between about 20%and about 70%, effective diffusion coefficient D is typically betweenabout 0.7×10⁻⁶ centimeters²/second and about 2.7×10⁻⁶centimeters²/second. Effective diffusion coefficient D for controlsolution typically is between about 4.0×10⁻⁶ centimeters²/second andabout 7.2×10⁻⁶ centimeters²/second.

Finally, test meter 600 displays an appropriate error message if thefluid sample is not in the acceptance range for bodily fluid or controlsolution, depending on which type of fluid sample has been applied totest strip 100, or allows the user to proceed with testing, as set forthby step 890.

Estimated effective diffusion coefficient D can also be used todistinguish between test strips 100 that include microelectrodes 120 andthose that do not include microelectrodes 120. Estimated effectivediffusion coefficient d may also be used to determine if reagent layer22 has been formulated or coated incorrectly. FIG. 20 is a flowchartillustrating a sequence of steps in a method 900 used by a test meter todetermine if a test strip 100 includes a plurality of microelectrodes120 and includes a reagent layer 22 that has been correctly formulatedand coated according to an exemplary embodiment.

Method 900 includes providing a test strip 100 with a referenceelectrode 10, a first working electrode 12, an optional second electrode14 and a test meter 600, as set forth by step 910. First workingelectrode 12 includes a plurality of microelectrodes 120 (i.e., amicroelectrode array 110) with each microelectrode 120 having a diameterbetween about 3 microns and about 50 microns and separated by about 5 toabout 10 times the diameter thereof. Reference electrode 10 includes asurface area that is at least equal to the surface area ofmicroelectrode array 110.

As set forth in step 920, the fluid sample is applied to test strip 100at t₀ and is allowed to react with reagent layer 22 for a reactionperiod t_(R) (see FIG. 14). The presence of sample in the reaction zoneof test strip 100 is determined by measuring the current flowing throughfirst working electrode 12. The beginning of reaction period t_(R) isdetermined to begin when the current flowing through first workingelectrode 12 reaches a desired value, typically about 0.150 nanoamperes(not shown), at which point a test voltage of zero millivolts is appliedbetween first working electrode 12 and reference electrode 10. Reactionperiod t_(R) is typically between about 2 and about 3 seconds and ismore typically about 2.5 seconds. After reaction period t_(R), the testvoltage in the exemplary method is applied to test strip 100 at t₁ for atotal test time t_(T). In an alternative method shown in FIG. 15, thereaction period t_(R) is omitted such that the start of the testcommences as soon as sufficient current is flowing through first workingelectrode 12.

As set forth in step 930, a limiting test voltage of about 100millivolts to about 600 millivolts, typically 400 millivolts, is appliedbetween reference electrode 10 and first working electrode 12 and testcurrent is measured as a function of time, as illustrated in FIG. 16. Asteady state current value I_(SS) is measured when an equilibriumcurrent value is attained, as set forth by step 940. Steady statecurrent value I_(SS) generally is reached between about 0.5 and about 2seconds after the test voltage is applied to test strip 100.

As set forth in step 950, the ratio of the test current to a steadystate current value

$\frac{I(t)}{I_{SS}}$

is then calculated for each time point at which the test current ismeasured. For microelectrode array 110 having a plurality of disk-shapedmicroelectrodes 120 where a limiting test voltage is applied, Equation 4above is used to estimate a ratio of the test current to thesteady-state current value.

Using Equation 4, effective diffusion coefficient D may be calculated byplotting the values

$\frac{I(t)}{I_{SS}}$

on the y-axis and

$\frac{1}{\sqrt{t}}$

on the x-axis as illustrated in FIG. 17 and set forth in step 960. Theresulting slope from the linear portion of the line may then becalculated and converted into effective diffusion coefficient D, as setforth in step 970.

Next, as set forth by step 980, a temperature-corrected diffusioncoefficient {tilde over (D)} is calculated by substituting effectivediffusion coefficient D into Equation 6 below that approximates thetemperature-dependent diffusion in a gel.

$\begin{matrix}{\overset{\sim}{D} = {{D\exp}\left\{ {\theta \left( {\frac{1}{T} - \frac{1}{T_{0}}} \right)} \right\}}} & (6)\end{matrix}$

Where:

-   -   {tilde over (D)} the temperature-corrected effective diffusion        coefficient in centimeter²/second;    -   D is the estimated effective diffusion coefficient in        centimeter²/second;    -   θ is a known constant for temperature-dependent diffusion;    -   T is temperature in Kelvin of the fluid sample as measured by        the test meter and generally is between about 283° K. and about        317° K.; and    -   T₀ is a reference temperature (e.g., room temperature) in        degrees Kelvin. T₀ ranges from about 293 degrees Kelvin to about        298 degrees Kelvin.

Next, test meter 600 determines if test strip 100 includesmicroelectrode array 110 and a correctly formulated and coated reagentlayer 22 by comparing a calculated value of temperature-correctedeffective diffusion coefficient {tilde over (D)} against an acceptancerange, as set forth by step 990. Temperature-corrected effectivediffusion coefficient {tilde over (D)} is typically about 1.8×10⁻⁶centimeters²/second and usually is between about 1.6×10⁻⁶centimeters²/second to 2.0×10⁻⁶ centimeters²/second.

Finally, if the calculated value for temperature-corrected effectivediffusion coefficient {tilde over (D)} is within the acceptance range,the user is allowed to proceed with testing. If, however, the calculatedvalue for temperature-corrected effective diffusion coefficient {tildeover (D)} is outside the acceptance range, test meter 600 displays anappropriate error message (e.g., the test strip was not recognized) tothe user, as set forth by step 995.

FIG. 21 is a plot illustrating simulated values fortemperature-corrected effective diffusion coefficient {tilde over (D)}for test strips 100 that include microelectrode array 110 and those thatdo not include microelectrode array 110. Arrowhead M indicates anappropriate range of values for temperature-corrected effectivediffusion coefficient {tilde over (D)} for test strip 100 that includemicroelectrode array 110. Values for temperature-corrected effectivediffusion coefficient {tilde over (D)} of test strips that do notinclude microelectrode array 110 would lie outside of arrowhead M asindicated by arrowheads N1 and N2 on this plot.

FIGS. 22 and 23 are perspective and side views, respectively, of anintegrated medical device 1000 that may include a plurality ofmicroelectrodes 120 according to exemplary embodiments. Integratedmedical device 1000 includes a test strip 1004 and a dermal tissuepenetration member 1002. Test strip 1004 has a reaction area 1005comprised of reagent layer 22 coated on reference electrode and one ormore working electrodes (not shown). Electrical contacts 1006 terminateon a proximal end 1010 of integrated medical device 1000 and are formedof any suitable conductive material, such as gold, silver, platinum orcarbon. Dermal tissue penetration member 1002 includes a lancet 1020adapted to pierce a user's skin and draw blood into reaction area 1005.Dermal tissue penetration member 1002 is adhered to test strip 1004 byan adhesive layer 1014. This adhesive layer 1014 can be heat seal orpressure sensitive adhesive. Lancet 1020 includes a lancet base 1022that terminates at the distal end 1012 of the assembled test strip.Further descriptions of integrated medical devices that may be used withthe present invention are in the U.S. patent application Ser. No.10/432,827 (published as 2004/0096959 on May 20, 2004) and U.S. patentapplication Ser. No. 10/143,399 (published as 2003/0143113 on Jul. 31,2003).

While the invention has been described in terms of particular variationsand illustrative figures, those of ordinary skill in the art willrecognize that the invention is not limited to the variations or figuresdescribed. In addition, where methods and steps described above indicatecertain events occurring in certain order, it is intended that certainsteps do not have to be performed in the order described but in anyorder as long as the steps allow the embodiments to function for theirintended purposes. Therefore, to the extent there are variations of theinvention, which are within the spirit of the disclosure or equivalentto the inventions found in the claims, it is the intent that this patentwill cover those variations as well.

1. A method of calculating a hematocrit-corrected glucose concentrationin a fluid sample, the method comprising: providing a test stripcomprising a reference electrode and a working electrode formed with aplurality of microelectrodes and coated with a reagent layer; applying afluid sample to the test strip for a reaction period; applying a testvoltage between the reference electrode and the working electrode;measuring a test current as a function of time; measuring a steady statecurrent value when the test current has reached an equilibrium;calculating a ratio of the test current to the steady state currentvalue; plotting the ratio of the test current to the steady statecurrent value as a function of the inverse square root of time;calculating an effective diffusion coefficient from the slope of thelinearly regressed plot of the ratio of the test current to the steadystate current value as a function of the inverse square root of time;and calculating a hematocrit-corrected concentration of analyte.
 2. Themethod of claim 1, wherein the calculating of the effective diffusioncoefficient step utilizes an equation of the form:$\frac{I(t)}{I_{SS}} = {1 + \left( \frac{2r_{d}}{\pi \sqrt{\pi \; {Dt}}} \right)}$where: I(t) is the current value in microamperes measured at time t;I_(SS) is the steady state current in microamperes; r_(d) is the radiusof a microelectode in centimeters; and t is time in seconds.
 3. Themethod of claim 2, wherein the calculating a hematocrit-correctedconcentration of analyte step further comprises: substituting theestimated diffusion coefficient into an equation of the form:$C_{red} = \frac{I_{S}}{4{nFDr}_{d}}$ where: C_(red) is a reducedmediator concentration; I_(SS) is the steady state current inmicroamperes; n is the number of electrons exchanged per ion thatundergoes an oxidation/reduction reaction; F is Faraday's constant; D isthe estimated diffusion coefficient in centimeter²/second; and r_(d) isthe radius of the microelectrode in centimeters; generating acalibration curve in which a y-axis is the reduced mediatorconcentration and an x-axis is a reference analyte concentration;calculating an analyte concentration by subtracting a calibrationintercept from the reduced mediator concentration and dividing with acalibration slope.
 4. The method of claim 1, wherein the test stripfurther comprises an insulation portion disposed on the plurality ofmicroelectrodes and the insulation portion has a height between aboutone micron and about six microns.
 5. The method of claim 1, wherein thereference electrode and the working electrode are comprised of gold. 6.The method of claim 1, wherein the reagent layer comprises an enzyme, amediator and a buffering agent wherein the mediator comprises ruthenium(III) hexamine.
 7. The method of claim 1, wherein the diameter of eachof the plurality of microelectrodes is between about 5 microns and about50 microns.
 8. The method of claim 1, wherein each of the plurality ofmicroelectrodes is spaced apart by a distance ranging from about 5 toabout 10 times the diameter of a microelectrode.
 9. The method of claim1, wherein each of the plurality of microelectrodes is spaced apart by adistance ranging from about 25 microns to about 500 microns.
 10. Themethod of claim 1, wherein the shape of each of the plurality ofmicroelectrodes is selected from a group consisting essentially of acircle, a square, a rectangle, an oval and an ellipse and combinationsthereof.
 11. A method of determining a type of fluid sample applied tothe test strip, the method comprising: providing a test strip having areference electrode and a working electrode, wherein the workingelectrode is formed with a plurality of microelectrodes and is coatedwith a reagent layer; applying a fluid sample to the test strip for areaction period; applying a test voltage between the reference electrodeand the working electrode; measuring a test current as a function oftime; measuring a steady state current value when the test current hasreached an equilibrium; calculating a ratio of the test current to thesteady state current value; plotting the ratio of the test current tothe steady state current value as a function of the inverse square rootof time; calculating an effective diffusion coefficient from the slopeof the linearly regressed plot of the ratio of the test current to thesteady state current value as a function of the inverse square root oftime; determining a type of a fluid sample applied to the test strip bycomparing a measured value for the effective diffusion coefficientagainst an acceptance range for a bodily fluid and a control solution;and displaying an appropriate error message if the effective diffusioncoefficient does not pass the acceptance range for the type of fluidsample applied to the test strip.
 12. The method of claim 11, whereinthe calculating of the effective diffusion coefficient step utilizes anequation of the form:$\frac{I(t)}{I_{SS}} = {1 + \left( \frac{2r_{d}}{\pi \sqrt{\pi \; {Dt}}} \right)}$where: I(t) is the current value in microamperes measured at time t;I_(SS) is the steady state current in microamperes; r_(d) is the radiusof a microelectode in centimeters; and t is time in seconds.
 13. Themethod of claim 12, wherein the calculating a hematocrit-correctedconcentration of analyte step further comprises: substituting theestimated diffusion coefficient into an equation of the form:$C_{red} = \frac{I_{S}}{4{nFDr}_{d}}$ where: C_(red) is a reducedmediator concentration; I_(SS) is the steady state current inmicroamperes; n is the number of electrons exchanged per ion thatundergoes an oxidation/reduction reaction; F is Faraday's constant; D isthe estimated diffusion coefficient in centimeter²/second; and r_(d) isthe radius of the microelectrode in centimeters; generating acalibration curve in which a y-axis is the reduced mediatorconcentration and an x-axis is a reference analyte concentration;calculating an analyte concentration by subtracting a calibrationintercept from the reduced mediator concentration and dividing with acalibration slope.
 14. The method of claim 11, wherein the test stripfurther comprises an insulation portion disposed on the plurality ofmicroelectrodes and the insulation portion has a height between aboutone micron and about six microns.
 15. The method of claim 11, whereinthe reference electrode and the working electrode are comprised of gold.16. The method of claim 11, wherein the reagent layer comprises anenzyme, a mediator and a buffering agent wherein the mediator comprisesruthenium (III) hexamine.
 17. The method of claim 11, wherein thediameter of each of the plurality of microelectrodes is between about 5microns and about 50 microns.
 18. The method of claim 11, wherein eachof the plurality of microelectrodes is spaced apart by a distanceranging from about 5 to about 10 times the diameter of a microelectrode.19. The method of claim 11, wherein each of the plurality ofmicroelectrodes is spaced apart by a distance ranging from about 25microns to about 500 microns.
 20. The method of claim 11, wherein theshape of each of the plurality of microelectrodes is selected from agroup consisting of a circle, a square, a rectangle, an oval and anellipse.
 21. A system comprising: a test strip having a referenceelectrode and a working electrode, wherein the working electrode formedwith a plurality of microelectrodes and coated with an reagent layer;and a test meter comprising: an electronic circuit for applying a testvoltage between the reference electrode and the working electrode; and asignal processor for measuring a test current and for calculating aneffective diffusion coefficient.
 22. The system of claim 21, wherein thetest strip further comprises an insulation portion disposed on theplurality of microelectrodes and the insulation portion has a heightbetween about one micron and about six microns.
 23. The system of claim21, wherein the effective diffusion coefficient is calculated from theslope of the linearly regressed plot of $\frac{I(t)}{I_{SS}}$ versus$\frac{1}{\sqrt{t}}$ based on an equation of the form:$\frac{I(t)}{I_{SS}} = {1 + \left( \frac{2r_{d}}{\pi \sqrt{\pi \; {Dt}}} \right)}$where: I(t) is the test current in microamperes measured at time t;I_(SS) is a steady state current in microamperes; r_(d) is a radius of amicroelectode in centimeters; and t is time in seconds.
 24. The systemof claim 23, wherein a hematocrit-corrected glucose concentration iscalculated by using the estimated diffusion coefficient and an equationof the form: $C_{red} = \frac{I_{S}}{4{nFDr}_{d}}$ where: C_(red) is areduced mediator concentration; I_(SS) is the steady state current inmicroamperes; n is the number of electrons exchanged per ion thatundergoes an oxidation/reduction reaction; F is Faraday's constant; D isa estimated diffusion coefficient in centimeter²/second; and r_(d) is aradius of the microelectrode in centimeters.
 25. The system of claim 34,wherein an effective diffusion coefficient is used to discriminatebetween a fluid sample of whole blood and a fluid sample of controlsolution by comparing the effective diffusion coefficient to anacceptance range for whole blood and an acceptance range for controlsolution and the effective diffusion coefficient is used to determine ifa test strip is formed with a plurality of microelectrodes bysubstituting the effective diffusion coefficient into an equation of theform:$\overset{\sim}{D} = {{D\exp}\left\{ {\theta \left( {\frac{1}{T} - \frac{1}{T_{0}}} \right)} \right\}}$Where: {tilde over (D)} is a temperature-corrected effective diffusioncoefficient in centimeter²/second; D is a estimated effective diffusioncoefficient in centimeter²/second; θ is a known constant fortemperature-dependent diffusion; T is a temperature in Kelvin of thefluid sample as measured by the test meter; and T₀ is a referencetemperature in degrees Kelvin.